Project Summary/Abstract The human retroviruses ? i.e., human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) ? infect over 37 million and 15 million people worldwide, respectively. HIV-1 causes acquired immunodeficiency syndrome (AIDS) and HTLV-1 infection causes fatal and debilitating diseases such as adult T cell leukemia/lymphoma (ATLL) and a variety of chronic inflammatory syndromes, most notably HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Differences in the basic mechanisms of HIV-1 and HTLV-1 replication can provide important insights into the molecular basis for viral pathogenicity and virus spread. For instance, HIV-1 can more efficiently spread and infect cells by cell-free infection, while HTLV-1 spread is strongly reliant upon cell-to-cell transmission as a means for virus spread. Virus eradication of lymphoid tissue has emphasized the importance of HIV-1 cell-to-cell transmission in HIV spread, which emphasizes the importance of parallel analyses with HTLV-1. An important open question in the field is the nature of retrovirus assembly in the context of cell-to-cell infection. This application proposes a comparative investigation of the mechanism of Gag and genomic RNA (gRNA) translocation to the plasma membrane in the context of non-polarized and polarized cells, which are implicated in cell-free and cell-to-cell virus spread, respectively. Gag translocation will be explored by examining Gag punctum biogenesis and mobility and by characterizing the dynamics of non-punctate Gag. Non-punctate Gag dynamics and Gag punctum biogenesis will be observed using total internal reflection fluorescence (TIRF) and super-resolution microscopy of Gag labeled with a photoconvertible fluorophore. Super-resolution microscopy will be used to investigate the dynamics of HIV-1 and HTLV-1 gRNA translocation to the plasma membrane in the absence and presence of wtGag or selected Gag mutants. Whether the translocation of gRNA to the VS occurs by directed movement in polarized cells will be determined, and whether Gag expression or Gag-gRNA interactions are required. The form of gRNA that is transported (i.e., monomer or dimer) will also be investigated. These studies will provide a deeper understanding of virus assembly in polarized cells, and provide new insights into the mechanisms of virus assembly at cell-cell contacts.